Fig 2: SDS-PAGE analysis of recombinant IL-18 before and after refolding.(A) SDS-PAGE showing soluble and insoluble fractions obtained after IL-18 expression in E. coli. Lane: M = protein marker, 1 = soluble fraction of wild-type (WT) IL-18, 2 = soluble fraction of mutant IL-18 DM, 3 = soluble fraction of mutant IL-18 DM1234, 4 = insoluble fraction of WT IL-18, 5 = insoluble fraction of mutant IL-18 DM, 6 = insoluble fraction of mutant IL-18 DM1234. (B) SDS-PAGE showing insoluble protein fractions after refolding. M = protein marker, 1 = IL-18 WT, 2 = IL-18 DM, 3 = IL-18 DM1234. (C) Western blot analysis of refolded IL-18 protein using anti-human IL-18 antibody. All samples were loaded onto 12% SDS-PAGE gel.

Fig 3: Acute arsenic treatment increases IL-1α, IL-1β and IL-18 mRNA levels as well as secretion levels in HaCaT cells. (A) HaCaT cells were treated with 0.5, 2 or 10 µM sodium arsenite for 24 h, or treated with 2 µM sodium arsenite for 24, 48 or 72 h. The mRNA levels of IL-1α, IL-1β and IL-18 in the cells were determined by real-time PCR. (B) The protein secretion levels of IL-1α, IL-1β and IL-18 in the media were determined by ELISA. (C) Cell viabilities were determined by cell counting kit-8. The experiments were repeated three times. Data are expressed as the mean ± SEM, *p < 0.05 vs. control.

Fig 4: Role of the Nrf2 antioxidant pathway in the inhibitory effect of Zn on HG-induced NLRP3 inflammasome activation in HPMCs. Nrf2 siRNA or scrambled siRNA (150 nM) were transfected into the HPMCs. Following transfection for 24 h, HPMCs were pretreated with 10 µM ZnSO4 or 1 µM TPEN for 24 h and then incubated with 126 mM glucose for 24 h. (A) Intracellular generation of ROS was detected using a DCFH-DA assay. The levels of (B) IL-1β and (C) IL-18 in the supernatants were determined by an ELISA assay. (D-F) NLRP3 inflammasome activity was examined using western blot analysis. (G and H) Activity of the Nrf2 antioxidant pathway was detected using western blot analysis. Values are presented as the mean ± standard deviation of three independent experiments. Student's two-tailed t-test was used to analyze significant differences. #P<0.05. HPMCs, human peritoneal mesothelial cells; Zn, zinc; HG, high glucose; TPEN, N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine; siRNA, small interfering RNA; ROS, reactive oxygen species; IL, interleukin; NLRP3, nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain-containing-3; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-related factor 2; Con, control.

Fig 5: Structural analysis of IL-18 based on MD simulations.(A) Structural alignment of wild-type (WT) and mutated IL-18. The binding site I is indicated in the figure. (B) Altered regions in IL-18 DM1234 structure. The interface between binding site I of each type of IL-18 and its IL-18 receptor α is shown. (C) Interaction of D35 residue of IL-18 WT and IL-18 DM1234 with V125 and S127 residues of IL-18 receptor α. (D) Interaction of D35 of IL-18 DM with V125 and S127 residues of IL-18 receptor α. The distance between interacting amino acids is indicated in angstrom (Å).